Emerging pandemic infections such as influenza A virus (IAV) and human immunodeficiency virus (HIV) remain major global health concerns due to high viral mutation rates, lack of defined immune correlates of protection and ineffective vaccines. CD4 T cells are essential in the immune response to viral infections because of their ability to enhance antibody production and help maintain CD8 T cell memory responses. Recently, a more direct role for CD4 cells during anti-viral responses has been appreciated, with many reports indicating CD4 T cells can acquire perforin mediated cytotoxicity that may be important for decreasing viral load. However, the signals that induce the differentiation of cytolytic CD4 cells (CD4 CTL) and the physiological impact of these cells in anti-viral immune responses remain obscure. The long-term goal of these studies is to understand the development of CD4 CTL and their role in vivo in order to inform vaccine strategies that target cell mediated immunity against infectious diseases. The objective of this proposal is to identify cytokine and transcription factor signals inducing CD4 CTL effector and memory responses leading to long term, protective anti- viral responses. Our central hypothesis is: class II restricted, perforin mediated cytotoxicity develops via IL-2 signaling and Th1 polarizing conditions, survives in a memory population and contributes to heterosubtypic protection. We formulated this hypothesis based on previous studies and preliminary data demonstrating that Th1 polarized CD4 effectors acquire perforin mediated cytotoxicity in response to IAV infection and a population of these cells can be identified at the memory stage. Further, IL-2 signaling via high affinity IL-2R pro- motes STAT5 phosphorylation and T-bet expression, correlating with GrB and perforin mediated cytotoxicity. This hypothesis will be tested by pursuing three specific aims: 1) Identify the cytokine signals required for full CD4 CTL development, 2) Identify the transcription factors driving CD4 CTL differentiation, and 3) Establish a role for CD4 CTL in the memory pool and recall responses to influenza infection. In the first aim, the role of IL-2 and inflammatory cytokine signals in CD4 CTL development will be established using cytokine and cytokine receptor knockout mice. In aim 2, candidate transcription factors such as STAT5, T-bet and Eomes, as well as unique transcription factors identified by microarray, will be tested for their ability to bind to the perforin promoter and control CD4 CTL differentiation. Finally, aim 3, will monitor the development of CD4 CTL memory responses and their ability to promote heterosubtypic immunity by using viruses that differ in outer coat proteins in CD8 deficient mice or using viruses that lack dominant CD8 epitopes. This research is innovative because it uses a unique in vitro activation model, as well as an in vivo approach to interrogate the requirements for differentiation of a novel subset of CD4 T cells with cytolytic activity. The proposed research is significant because it is the first step in understanding the mechanisms that drive CD4 CTL differentiation that has the potential to inform novel vaccine strategies targeting T cell immunity against emerging pathogens.